Under hypercontractile conditions associated with increased cell calcium, males show enhanced myocardial ischemia/reperfusion injury compared to females. Our aim was to identify the specific estrogen receptor involved and the mechanism responsible for this gender difference. Following brief treatment with isoproterenol, hearts were subjected to ischemia and reperfusion and postischemic contractile function and infarct size were measured in wild-type (WT) male and female mouse hearts, and female hearts lacking functional alpha estrogen receptor (aERKO), or the beta estrogen receptor (bERKO). WT males exhibited significantly poorer functional recovery and larger infarcts than WT females. aERKO females had ischemia-reperfusion injury similar to that observed in WT females; however,infarcts in bERKO females were significantly larger and recovery of function was poorer than WT or aERKO females, but similar to those seen in WT males. These data suggest that estrogen through the beta-estrogen receptor plays a role in the protection observed in the female heart. We also examined whether there were gender differences in sarcoplasmic reticulum (SR) Ca2+. We used the NMR Ca2+ indicator TFBAPTA to measure SR Ca2+ in perfused rabbit hearts. Addition of isoproterenol significantly increased SR Ca2+ in males from a baseline value of 1.13 mM to 1.52 mM. Female hearts had a basal SR Ca2+ that was not significantly different than males (1.04 mM), and addition of isoproterenol to females resulted in a time-average SR Ca2+ (0.97 mM) that was significantly less than in males. To confirm this gender difference in SR Ca2+ after isoproterenol, we measured caffeine-induced release of SR Ca2+ with fura-2 in isolated ventricular myocytes. Ca2+ release after caffeine addition in untreated male myocytes was 399 nM and significantly increased to 612 nM in isoproterenol treated myocytes. Ca2+ release after caffeine addition in untreated females was 360 nM and increased to 485 nM with isoproterenol treatment. There was a significant difference in SR Ca2+ release between male and female myocytes treated with isoproterenol. Treatment of female myocytes with L-NAME, an inhibitor of nitric oxide synthase resulted in an SR Ca2+ release that was higher than that measured in females treated only with isoproterenol and not significantly different than the SR Ca2+ release measured in males with isoproterenol. This gender difference in SR Ca2+ handling may contribute to reduced ischemia-reperfusion injury observed in females. We have also found that intracellular sodium ([Na]) plays an important role in Ca overload. To explore the role of [Na] in the gender difference in I/R injury, we measured [Na] using 23Na nuclear magnetic resonance spectroscopy with a shift reagent (Tm(DOTP)5-) in isolated perfused mouse hearts. Contractile function was measured simultaneously through a balloon in the left ventricle connected to a pressure transducer. At baseline there was no significant gender difference in [Na] (Male: 15.8 mM vs. Female: 16.6). During 20 minutes of ischemia, [Na] increased to 197% of pre-ischemic level in male hearts, whereas [Na] accumulation in female hearts was slightly but significantly less (176%) than in males. After brief (1 min) treatment with isoproterenol (ISO, 10nM) prior to ischemia, male hearts exhibited poorer recovery of post-ischemic contractile function vs. female. Also, ISO-treated male hearts had a larger increase in [Na] during ischemia (216%) compared to that in female hearts (171%). We examined the role of nitric oxide in the gender difference in [Na]. The NO synthase inhibitor, L-NAME abolished the gender difference in [Na] during ischemia (Male: 204% vs. Female 200%) and also abolished the difference in functional recovery after reperfusion (Male: 17% vs. Female: 22%), suggesting that gender differences in [Na] regulation during ischemia are NO-dependent. In conclusion, the female heart has improved [Na] homeostasis during ischemia through an NO-dependent pathway. Previous epidemiological studies have indicated that pre-menopausal females are at a decreased risk for developing cardiovascular disease compared to males of the same age. To study gender differences in the development and progression of pressure overload hypertrophy, we performed transverse aortic constriction (TAC) and sham operations in male and female mice. Body, heart, and lung weights were measured 2 weeks post surgery. TAC produced a significant increase in heart to body weight ratio compared to sham operation for both sexes. TAC female mice showed a 31% increase in heart to body weight ratio compared to sham. TAC male mice showed a 64% increase in this ratio compared to sham, which was significantly greater than their female counterparts (p = .017). Lung to body weight ratio, an indication of left ventricular failure, also significantly increased in TAC male mice compared to sham (p < .01). In TAC female mice, this ratio remained unchanged compared to sham. These differences prompted us to investigate gender associated gene profiles in the heart through a series of oligonucleotide microarray experiments. Hypertrophied TAC male vs female hearts as well as sham male vs female hearts were compared to identify genes differentially expressed. Genes up-regulated or down-regulated were confirmed with real time-PCR. Several genes potentially crucial to cardiovascular function including lipoprotein lipase, glutathione-S-transferase (GST) mu1, and transforming growth factor -3 were differentially expressed between TAC males and females. In order to establish the importance of the alpha versus the beta estrogen receptor in mediating the attenuation of pressure overload hypertrophy, alpha estrogen receptor knock out (aERKO) and beta ERKO (bERKO) mice were subjected to TAC and sham surgery. aERKO females showed a degree of hypertrophy nearly identical to that seen in wild type females, indicating the alpha estrogen receptor is not essential for the attenuation of hypertrophy observed in wild type females. In contrast, bERKO females had increased hypertrophy, similar to that observed in males and significantly less than observed in WT and aERKO females. In conclusion, these experiments indicate female mediated cardioprotection in the setting of pressure overload hypertrophy is mediated by the beta estrogen receptor. Microarray analysis confirms distinct gender associated expression profiles underlie the phenotypic difference.